MiR-322/424 and -503 Are Induced during Muscle Differentiation and Promote Cell Cycle Quiescence and Differentiation by Down-Regulation of Cdc25A

Cell Culture

Human embryonic kidney (HEK)-293T and U2OS (osteosarcoma) cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% of fetal bovine serum and 1% penicillin/streptomycin. HCT116 and HCT116 ^p21−/− (colon cancer) cells were maintained in McCoy's 5A media (Invitrogen) supplemented with 10% of fetal bovine serum and 1% penicillin/streptomycin. C2C12 mouse skeletal myoblasts (American Type Culture Collection; Yaffe and Saxel, 1977) were maintained in DMEM supplemented with 20% fetal calf serum (GM) and 1% penicillin/streptomycin. Myogenic differentiation (Andres and Walsh, 1996) into myotubes was induced by changing subconfluent cells to DMEM containing 2% heat-inactivated horse serum (DM).

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Cells were collected at different days of differentiation and total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RT-PCR for microRNA was performed using Ncode miRNA first-strand cDNA synthesis and qRT-PCR kit (Invitrogen). cDNA synthesis for mRNA detection was carried out using SuperScript III first-strand synthesis system for RT-PCR (Invitrogen). mRNA was detected by quantitative (q)PCR using SYBR Green PCR master mix in an ABI cycler and quantified with ABI 7300 software (Applied Biosystems, Foster City, CA).

Luciferase Reporter Assays

Human and mouse Cdc25A 3′ untranslated regions (UTRs) (41-1000 bp and 51-1369 bp, respectively) were amplified from cDNA and cloned into modified pRL-CMV (MCS) described previously (Kim et al., 2006) by using EcoRI and XhoI restriction sites. U2OS cells were transfected with microRNAs using RNAiMAX transfection reagent (Invitrogen). After 48 h, luciferase plasmids were transfected using Lipofectamine 2000 (Invitrogen), pGL3 control vector (Promega, Madison, WI) was transfected as internal control. At 24 h after plasmid transfection, luciferase assays were performed with Dual-Luciferase Reporter Assay system (Promega) per the manufacturer's instructions. Luminescent signal was quantified by luminometer (Monolight 3020; BD Biosciences, San Jose, CA). Each value from Renilla luciferase construct (Rr) was first normalized to the firefly (Photinus pyralis) luciferase assay value (Pp) from the cotransfected pGL3-control vector (Promega). Each Rr/Pp value in the microRNA transfections was again normalized to the Rr/Pp value obtained in control small interfering RNA (siRNA)-transfected cells.

Plasmid Construction

Cdk2-wild type (WT) and cdk2-AF were subcloned to pMSCV retroviral vector using EcoRI/NotI site; Cdc25A and mutants were subcloned to either pLPCX or pMSCV retroviral vectors using BamHI/NotI site. Retrovirus was made in HEK-293T cells cotransfected with virus packaging plasmids.

Plasmid, siRNA, and microRNA Mimic and Antisense Transfection

Plasmids were transfected using Lipofectamine 2000 transfection reagent (Invitrogen) siRNA, and microRNA mimics were transfected into U2OS or HCT116 ^p21−/− using RNAiMAX (Invitrogen) as per manufacturer's protocol. The antisense oligonucleotides (locked nucleic acid or 2′O-methyl) were transfected into C2C12 at 0 and 48 h in serum-containing medium. At 72 h cells were differentiated by serum depletion (day 0 in Figures 1C or 5E).

Western Blotting and Antibodies

For Western blotting, cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, and 10% glycerol) supplemented with protease inhibitor mix (Sigma-Aldrich, St. Louis, MO). Unless otherwise mentioned, 30 μg of protein was resolved by SDS-polyacrylamide gel electrophoresis, transferred, and immunoblotted with various antibodies. The antibodies used were anti-p21 (C-19), anti-p27 (C-19), anti-Cdk2 (M2), anti-cyclin E (HE111), anti-cyclin A (H-432), anti-Cdc25A (F-6), anti-Wee1 (H-300) (all from Santa Cruz Biotechnology, Santa Cruz, CA); and anti-Cdc2-phospho-Tyr15 (Cell Signaling Technology, Danvers, MA). Mouse monoclonal antibody anti-myosin heavy chain (MHC), anti-myogenin, and anti-β-actin antibody were from Sigma-Aldrich.

Fluorescence-activated Cell Sorting (FACS) Analysis

Cells were harvested by trypsinization and then fixed with 70% ethanol for 24 h at 4°C. Fixed cells were stained in 1 ml of propidium iodide solution (0.05% NP-40, 50 μg/ml propidium iodide, and 10 μg/ml RNase A) at least for 2 h at 4°C. Stained cells were analyzed with a flow cytometer using CellQuest software (both from BD Biosciences).

Immunoprecipitation and cdk2 Kinase Assays

For total cdk2, cyclin E- or cyclin A-associated kinase assays 100 μg of protein was immunoprecipitated and pulled down on G-Sepharose beads. The beads were washed three times with lysis buffer and twice with kinase buffer (50 mM HEPES-NaOH, pH 7.4, and 25 mM MgCl₂). The beads were then incubated with 25 μl of kinase reaction mixture (50 mM HEPES-NaOH, pH 7.4, 25 mM MgCl₂, 0.5 mM dithiothreitol, 50 μM ATP, 5 μCi of [γ-³²P]ATP, and 2 μg of histone H1) at 30°C for 30 min. The reaction was stopped by the addition of 12.5 μl of 3× SDS sample buffer. Samples were resolved by SDS-PAGE, and the gel was dried before autoradiography.

MiR-322 and -503 Induced during Muscle Differentiation Are Required for cdk2 Inhibition

On depletion of serum, proliferating mouse C2C12 myoblasts stop proliferation and differentiate in 1–3 d into myotubes that are positive for MHC and myogenin (Kim et al., 2006). Propidium iodide staining of nuclei for DNA content and flow cytometry for the distribution of cells in different phases of the cell cycle revealed that the G1 population increased and the S and G2/M population decreased concurrently (Figure 1A and Supplemental Figure S1A). Quantitative RT-PCR was used to screen twenty microRNAs known to have cell cycle targets in these differentiating myoblasts. The screen revealed that two microRNAs, miR-322 and miR-503, were increased during this differentiation process (Figure 1B).

Figure 1.

miR-322 and -503 are induced during the differentiation of C2C12 myoblasts, required for optimal inhibition of cdk2 kinase and accelerate expression of myogemic markers. (A) Increase in G1 population cells during differentiation. Days of serum deprivation are indicated. The results are expressed as percentage of change of cells in a given phase of the cell cycle in differentiating cells relative to undifferentiated C2C12. The primary FACS results are in Supplemental Figure S1. (B) Quantitative RT-PCR of the miR-322 and -503 during differentiation of C2C12 myoblasts. Days of serum deprivation are indicated. Fold changes of these microRNAs, normalized to U6 sRNA, are expressed relative to day 0 of serum depletion. (C) Left, total cdk2 was immunoprecipitated by anti-cdk2 antibody from C2C12 cells transfected with locked nucleic acid antisense to miR-322 and -503, and cdk2 kinase activity measured in vitro on histone H1. Autoradiogram of phospho-H1 is shown. Western for cdk2 shows amounts of the kinase in the input lysates. Day 0: 72 h after first anti-sense transfection and harvested before adding differentiation medium. Days of differentiation are indicated. Right, quantification of the H1 kinase activity (normalized to cdk2 quantity) is shown. The results are expressed relative to normalized kinase activity on day 0. (D and E) Transfection of miR-322 and -503 accelerate expression of myogenic markers MHC and myogenin. Fold change in the expression of MHC (D) and myogenin (E) mRNA compared with control siRNA-treated cells are shown. Days of differentiation are indicated.

The cdk2 kinase activity of C2C12 cells undergoing differentiation was followed by immunoprecipitating cdk2 and assaying kinase activity on histone H1 (Figure 1C, left). In control antisense-transfected cells (same as untransfected cells), the H1 kinase activity declined rapidly by 1–2 d of differentiation. To ascertain whether miR-322 and -503 had any role in the cdk2 repression, in parallel experiments we transfected C2C12 cells with locked nucleic acid (LNA) antisense oligonucleotides to miR-322 and -503. The anti-microRNA oligonucleotides (LNA) caused the kinase activity of cdk2 to persist up to day 3 of the differentiation (compare lanes 3 and 4, 5 and 6, and 7 and 8). Quantification of this Western blot (Figure 1C, left) revealed that H1 kinase activity (normalized to input) was higher in cells with anti-microRNA oligonucleotides in differentiation medium (Figure 1C, right). We thus hypothesized that the induction of miR-322 and -503 contributed to the cdk2 inhibition during myotube differentiation.

We next tested whether these microRNAs accelerated muscle differentiation. Transfection of synthetic RNA duplexes mimicking the miR-322 and -503 in C2C12 cells indeed accelerated myogenic differentiation as determined by the induction of MHC and myogenin mRNA (Figure 1, D and E; and Supplemental Figure S2). Reciprocally inhibition of miR-322 and -503 using 2-O′-methyl antisense oligonucleotides decreased expression of myogenin and MHC mRNA (Supplemental Figure S3).

MiR-322/424 and -503 Promote G1 Arrest in Osteosarcoma Cells

To determine whether these microRNAs could induce a G1 block in another cell lineage, we transfected synthetic RNA duplexes mimicking the microRNAs in rapidly proliferating cell cycle-asynchronous human U2OS osteosarcoma cells. Transfection of miR-424 and miR-503 independently increased the G1 and decreased the S and G2/M population of cells (Figure 2A and Supplemental Figure S1B).

Figure 2.

The G1 accumulation and cdk2 inhibition upon miR-424 or miR-503 transfection in U2OS and HCT116 cells are independent of p21 and p27. (A) Change in cell cycle stage after miR-424 and -503 were transfected in U2OS cells. DNA contents were measured after 72 h, and the percentage of change in microRNA-treated cells relative to control siRNA treated cells was plotted. Mean ± SD of at least three independent experiments. Primary FACS results are in Supplemental Figure S1. (B) U2OS cells collected in mitosis by nocodazole and shake-off were put into fresh medium, transfected with control or microRNA mimic and aphidicolin added to the medium to hold cells in G1/S. Twenty four hours later, cells were released from aphidicolin block and placed in nocodazole to trap cells moving from S phase to mitosis. FACS analysis for DNA content at indicated time points after release from aphidicolin. (C) Top, protein lysate were extracted from U2OS cells 72 h after transfection of sicontrol (GL2), miR-424, and -503. Western blot analysis for anti-Rb shows hypophosphorylated (faster migrating) Rb protein in cells transfected with the microRNAs. Bottom, quantification of the hypophosphorylated Rb normalized to total Rb. (D) Total and cyclin A-associated cdk2 were immunoprecipitated (IP), and kinase assays performed as mentioned in Materials and Methods (H1: autoradiogram of phospho-H1). Five percent input and corresponding IP for cdk2 or cyclin A were immunoblotted for cdk2 as loading controls. (E) Immunoblot with indicated antibodies of lysates extracted from U2OS cells transfected with indicated RNA duplexes. Loading control, nonspecific band (*) in the anti-p21 blot and β-actin in the anti-p27 blot. (F) Change in percentage of HCT116 ^p21−/− cells in different phases of cell cycle upon overexpression of miR-424 or -503 relative to control RNA duplex. Mean ± SD of at least three independent experiments. Primary FACS results are in Supplemental Figure S1. (G) Same as in F except that p27 was initially depleted by si-p27 (see Supplemental Figure S1D) before transfecting miR-424 and -503. Primary FACS results are in Supplemental Figure S1. (H) Cdk2 kinase activity of HCT116 ^p21−/− cells depleted of p27 and overexpressing miR-424 or -503.

Unless mentioned specifically, we transfected 75 nM of miR-322/424 or 25 nM of miR-503. Quantitative RT-PCR showed similar levels of miR-322 and -503 in C2C12 cells on day 3 of differentiation and in microRNA transfected U2OS cells (Supplemental Figure S4).

To further establish whether these microRNAs caused a G1 accumulation, U2OS cells were synchronized in mitosis by nocodazole block and shake off and then transfected with either negative control GL2 duplex or the microRNA mimics as the cells transited through G1 and accumulated at the G1/S transition because of exposure to aphidicolin, an inhibitor of the replicative DNA polymerase. After giving the microRNAs ∼18 h to exert their effect, the cells were released from the aphidicolin block (0 h) into a new nocodazole block, and their progression through S phase and G2 was followed by propidium iodide FACS (Figure 2B). Ninety percent of the cells transfected with the negative control duplex moved through S and G2 and accumulated in mitosis with a G2 content of DNA at 10–24 h after release from aphidicolin. In contrast, nearly 50% of the cells transfected with miR-424 or miR-503 remained stuck at the G1-S boundary. Thus, the two microRNAs block the progression of cells into S phase of the cell cycle.

We then focused on the cell cycle proteins that are important for progression from G1 to S. Rb is hyperphosphorylated and migrates in SDS-PAGE as a slower moving band upon phosphorylation by cdk2 at the G1–S transition. Consistent with miR-424 and -503 inhibiting this transition, a faster moving, hypophosphorylated Rb band was visible in the U2OS cells transfected with these microRNAs (Figure 2C, top). This hypophosphorylation was more pronounced in miR-424– and -503–transfected lanes compared with sicontrol lane as determined by quantification of this Western blot (Figure 2C, bottom). When cell lysates were immunoprecipitated by antibodies to cdk2 or cyclin A, although equal amounts of cdk2 protein were precipitated from all three lysates, the kinase activity in vitro on histone H1 was significantly attenuated in miR-424– or -503–transfected cells (Figure 2D). Cyclin A level went down in these samples (Supplemental Figure S5). Therefore, to immunoprecipitate equal amount of Cyclin A we used more cell lysate in miR-424– and -503–treated samples compared with sicontrol-treated sample. Thus, transfection of either microRNA inhibits cdk2 kinase activity, perhaps suggesting how they might interfere with G1–S transition.

Cdk2 Inhibitors Are Induced but Dispensable for the G1 Arrest

Transfection of miR-424 or -503 into U2OS cells induced the cdk inhibitors p21 and p27 (Figure 2E). However, because degradation of both the inhibitors is dependent on active cdk2 kinase, their induction could be secondary to cdk2 inhibition from a different proximate cause. Indeed, even after double knockdown of p21 and p27 in U2OS cells, transfection of miR-424- or of -503 still caused an accumulation of the cells in the G1 phase of the cell cycle and inhibition of cdk2 kinase activity (Supplemental Figures S1K and S6, A–C).

To independently examine whether p21 or p27 were necessary for the G1 arrest by miR-424 or -503, we turned to HCT116 colon cancer cells that had been engineered by homologous recombination to be null for the p21 locus (HCT116 ^p21−/−) (Waldman et al., 1995). Mir-424 or -503 transfection increased the G1 population and decreased the post-G1 population of cell (Figure 2F and Supplemental Figure S1C). We repeated the experiment after knocking down p27 by siRNA (Supplemental Figure S6D). Despite the fact that p21 was absent and p27 had been virtually eliminated, transfection of miR-424 or -503 still increased the G1 population and decreased the percentage of cells in the later stages of the cell cycle (Figure 2G and Supplemental Figure S1D). In vitro kinase assays with immunoprecipitates of cdk2 or of cyclin E or A revealed that the microRNAs continued to decrease cdk2 kinase activity despite the absence of p21 and decrease of p27 (Figure 2H). We concluded that cdk2 kinase was being inhibited by the microRNAs by a mechanism independent of the cdk2 inhibitors.

Cdc25A Is Repressed by miR-424 and -503 in Human Cancer Cell Lines

The microRNA target prediction algorithms suggested that cyclins D and E and Cdc25A are potential targets of these two microRNAs. Levels of cyclin D or E were unchanged in the cells transfected with the microRNAs (data not shown). In contrast, immunoblots of extracts of U2OS cells show that transfection of miR-424 or -503 represses the Cdc25A protein and mRNA levels (Figure 3A and Supplemental Figure S7). Identical results were obtained in HCT116 ^p21−/− cells with a knockdown of p27 (Figure 3B). These results are consistent with the notion that the microRNAs target Cdc25A.

Figure 3.

miR-424 and miR-503 down-regulate Cdc25A. (A) Western blot for Cdc25A and tubulin (loading control) on lysate from U2OS cells transfected with indicated RNA duplexes. Right, HCT116 cells were treated with UV, and the degradation of Cdc25A used to indicate specificity of the immunoblot signal. (B) Same as in A except HCT116 ^p21−/− cells were depleted of p27 protein by siRNA against p27 and indicated RNA duplexes transfected. (C) Western blot analysis of Cdc25A protein in C2C12 cells at indicated times after serum deprivation. Anti-tubulin is shown as loading control. Dilutions of the day 0 extract are loaded in the first three lanes to show the signal expected when Cdc25A protein is 50% the level in day 0. (D) Real-time qRT-PCR of mRNA at indicated times upon differentiation. Cdc25A mRNA level was plotted after normalizing to that of β-actin.

Cdc25A Protein Is Decreased Posttranscriptionally during Differentiation of C2C12 Myoblasts

When C2C12 cells are induced to differentiate by serum depletion, Cdc25A protein decreased to levels below detection within 2 d (Figure 3C). Interestingly, qRT-PCR of mRNA reveals that Cdc25A mRNA declines in a more gradual manner, with a 50% reduction on day 1 that is then repressed even further on day 3 of serum depletion (Figure 3D). The faster kinetics of repression of Cdc25A protein relative to that of the RNA suggests that posttranscriptional mechanisms are at play in inhibiting Cdc25A during muscle differentiation. This, too, is consistent with the idea that muscle differentiation-induced microRNAs may target Cdc25A during differentiation.

Cdc25A Is a Direct Target of the Two MicroRNAs

The 3′UTR of both human and mouse Cdc25A had sequences that could anneal to the seed sequences of miR-424/322 or -503 (Figure 4A). A luciferase reporter plasmid with the 3′UTR of human Cdc25A was repressed by cotransfection of miR-503 (Figure 4B) or miR-424 (Figure 4C). Mutation of both the putative target sequences for miR-503 attenuated the repression by the microRNA (Figure 4B), demonstrating that we have identified the relevant target sites for this microRNA. In contrast, mutation of the putative target site for miR-424 did not reprieve the repression (Figure 4C), suggesting that miR-424 may act through a nonseed matching sequence in the 3′UTR that we have not yet identified. Such nonseed match mediated repression of targets by microRNAs has been described previously (Lal et al., 2009).

Figure 4.

miR-424 and -503 directly target Cdc25A. (A) Two predicted target sites of miR-424/322 and -503 in the 3′UTR of human and mouse Cdc25A. Mutant 1, mutant 2 (in human Cdc25A) and mutant 3 (in mouse Cdc25A) are indicated. (B–E) Luciferase assays were performed to measure the effect of miR-424/322 and -503 on a Renilla luciferase reporter fused to a perfect match to the microRNA (pRL-503 or pRL-424) or to human or mouse Cdc25A 3′UTR (see Materials and Methods). The results are expressed as Renilla luciferase activity relative to the firefly luciferase expressed from a cotransfected plasmid and serving as a transfection control. The ratio seen in control duplex transfected cells is 1. (B) Luciferase with human Cdc25A 3′UTR repressed by miR-503 mimic. (C) Same reporter as in B repressed by miR-424 mimic. (D) Luciferase with mouse Cdc25A 3′UTR repressed by miR-503 mimic. (E) Same as in D, except with miR-322 mimic. Because the differences were small in E, the p values in a Fisher's test are shown to indicate statistical significance.

The results were reinforced by analogous effects of miR-503 and miR-322 on a luciferase reporter with the 3′UTR of mouse Cdc25A (Figure 4, D and E). In both these cases, mutation of the putative target sites attenuated the repression by the microRNA. Despite the extensive variation in the sequences of the 3′UTRs of human and mouse Cdc25A, the evolutionary conservation of repression by both the microRNAs suggested that repression of Cdc25A by these microRNAs could be developmentally important.

Overexpression of Cdc25A Delays Differentiation but Also Reveals That the Protein Is Degraded by Proteasomes during Differentiation

To test whether the decrease in Cdc25A is important for the normal kinetics of differentiation of C2C12 myoblasts to myotubes, we established C2C12 derivatives infected with either an empty retroviral vector (vector) or a retrovirus overexpressing Cdc25A from its open reading frame alone. The latter construct is missing the 3′UTR of Cdc25A. When these cells were induced to differentiate by serum depletion, the appearance of myogenin, a differentiation marker, was significantly delayed in the C2C12 cells overexpressing Cdc25A (Figure 5A). This result is consistent with the hypothesis that repression of Cdc25A is an important element of the differentiation program.

Figure 5.

Ectopic expression of Cdc25A delays differentiation, although Cdc25A protein is still degraded in a proteasome-dependent manner. (A) C2C12 cells stably infected with either empty retroviral vector or retrovirus expressing Cdc25A were differentiated and assayed for induction of myogenin, a differentiation marker on indicated days after serum deprivation. Anti-actin is shown as loading control. (B) Lysates from the experiment in A were immunoblotted for anti-Cdc25A. 100 μg of total protein from vector-infected cells (endogenous Cdc25A) and 30 μg from Cdc25A-overexpressing cells (exogenous Cdc25A) were resolved. (C) Cdc25A overexpressing C2C12 cells were differentiated by serum deprivation. Cells were treated with or without proteasome inhibitor MG-132 (5 μg/ml) for 4 h before harvest at indicated times. Cells were lysed and immunoblotted for anti-Cdc25A. The rest are as described in B. (D) Cdc25A-WT and Cdc25A-2A were stably expressed in C2C12 cells, which were then differentiated, harvested at indicated times, lysed, and immunoblotted from Cdc25A. (B–D) Anti-tubulin blots serve as loading control. (E) C2C12 myoblasts were preloaded with 2′-O-methyl oligonucleotide antisense to either control RNA (luciferase) or a mixture of anti-sense to miR-322 and -503 and then induced to differentiate. Day 0, first day of serum deprivation, which is 72 h after first antisense transfection. Cells were treated with MG-132 as described in C, and 100 μg of cell lysate was immunoblotted for Cdc25A and tubulin (loading control).

Immunoblots of the C2C12 cells indicated that, consistent with Figure 3C, the endogenous Cdc25A protein decreased by day 1 of serum depletion (Figure 5B). The overexpressed exogenous Cdc25A persisted until day 3 of differentiation. Surprisingly, however, although the exogenous Cdc25A was expressed from a heterologous promoter and did not have a 3′UTR that could be targeted by muscle-differentiation-induced microRNAs, the protein still decreased during differentiation. Cdc25A is ubiqutinylated by multiple pathways and is targeted for proteasome-mediated proteolysis following DNA damage. This made us wonder whether proteasome-mediated degradation was also important for Cdc25A decrease during differentiation. Indeed, the addition of MG132, an inhibitor of proteasomes, increased both endogenous and exogenous Cdc25A in C2C12 myoblasts (Figure 5C). When the myoblasts were induced to differentiate by serum depletion, it was interesting to note that the endogenous Cdc25A protein was undetectable even after MG132 treatment after day 2 of differentiation, suggesting that nonproteasome mediated pathways (such as microRNAs and transcriptional repression) come into effect to keep Cdc25A repressed during differentiation. The proteasome-mediated degradation, however, continues to be active, because the exogenous Cdc25A protein is readily induced even after 3 d of differentiation when the proteasomes are inhibited by MG132.

S82 in the DSG motif of Cdc25A is phosphorylated by Chk1 to promote its degradation by proteasomes after DNA damage (Kanemori et al., 2005). This site and a related DDG motif are recognized by the βTRCP subunit of CRL1^βTRCP for polyubiquitination. To test whether these two sites were relevant for Cdc25A degradation during muscle differentiation, we used C2C12 cells overexpressing wild-type Cdc25A or Cdc25A-2A mutated at the two sites (S82A and D220A) (Figure 5D, which is a much lighter exposure than Figure 5B). Compared with the wild-type exogenous Cdc25A, the mutant exogenous protein was more stable during the earlier stages of differentiation, but still disappeared on day 3 of the differentiation regime. MG132 treatment revealed that the disappearance of Cdc25A-2A on day 3 was still dependent on proteasomes (data not shown), suggesting that another ubiquitylation pathway becomes active after three days of serum depletion to target Cdc25A protein for degradation.

Antisense to miR-322 and -503 Sustains Cdc25A Levels in Differentiating C2C12 Myoblasts

We next tested whether antisense to the two microRNAs will slow down the rate of disappearance of Cdc25A during differentiation. A control antisense 2′O-methyl oligonucleotide or a mixture of antisense to miR-322 and -503 was transfected into C2C12 myoblasts, which were then induced to differentiate by serum depletion (Figure 5E). Because Cdc25A is also targeted by proteasomes during differentiation, we neutralized the proteasome activity by treating the cells for 4 h with MG132 before harvest. On days 0 and 1, the anti-miR-322 and -503 did not seem to increase Cdc25A protein level relative to control anti-sense RNA even in the presence of MG132. On days 2 and 3 of differentiation (compare lane 10–12 and 14–16), however, transfection of the anti-miR oligonucleotides caused a detectable persistence of Cdc25A protein relative to the control oligonucleotide when the proteasomes were inhibited by MG132. Note that transfection of antisense to 2′-O-methyl oligonucleotides to microRNAs is an imperfect tool for knocking down microRNA activity.

Thus, despite the fact that Cdc25A (and most likely other cdk2 regulators) are regulated by multiple microRNA- and nonmicroRNA-dependent pathways, neutralization of miR-322 and -503 by imperfect tools showed lesser repression of Cdc25A (Figure 5E) and greater persistence of cdk2 kinase activity (Figure 1C) in the differentiating myoblasts.

Inhibitory Phosphorylation on cdk2 T14 and Y15 Is Important for the Differentiation of C2C12 Myoblasts

We next investigated whether Y15 on cdk2 is phosphorylated in the myoblasts. After immunoprecipitating with anti-cdk-phosphotyrosine15 antibody, immunoblot with anti-cdk2 revealed a weak but detectable phosphotyrosine signal on endogenous cdk2 in lysates of C2C12 on days 0, 1, and 2 of serum depletion (Figure 6A, top). Reasoning that the signal was weak because of rate-limiting quantities of cdk2, we overexpressed exogenous FLAG-tagged wild-type cdk2 in C2C12 and then measured phosphoY15 levels in these cells as they differentiated. A clear phosphotyrosine signal was detected on cdk2 at days 0, 1, and 2 of differentiation. Cdk2 paired with a cyclin is the preferential substrate of the Wee1 kinase that phosphorylates cdk2 on Y15. The disappearance of the phosphoY15 signal at day 3 made us wonder whether this was due to a decrease in partner cyclins that are transcriptionally down-regulated during differentiation. Indeed, when we immunoprecipitated cyclin A from the C2C12 cells and exclusively examined the cdk2 that was associated with cyclin A, the phosphorylation on tyrosine was discernible on all the days examined (Figure 6A, bottom). The failure to see an increase in phospho-tyrosine during the differentiation process despite the loss of Cdc25A made us wonder whether the kinase that phosphorylates the tyrosine, Wee1, was decreasing concurrently. Indeed the Wee1 kinase decreased upon serum depletion (Figure 6B). The persistence of the inhibitory phosphorylation during the differentiation program despite the loss of Wee1, could only be explained by the decrease in Cdc25A.

Figure 6.

Cdk2-AF mutation delays differentiation of C2C12 cells. (A) Cdk2 is phosphorylated on tyrosine in differentiating C2C12 cells. C2C12 cells infected with empty retroviral vector or retrovirus expressing FLAG-tagged cdk2-WT were differentiated. Top, cell lysates at indicated times were immunoprecipitated with anti-cdc2-phosphotyrosine15 antibody and immunoblotted for cdk2. Bottom, cell lysates were immunoprecipitated with anti-cyclin A antibody and the tyr15 phosphorylation of cyclin A-associated cdk2 assessed as in the top panel. (B) Western blot analysis of Wee1 protein in C2C12 cells at indicated times after serum deprivation. Anti-tubulin serves as loading control. (C) Overexpression of cdk2-AF mutant represses G1 block in differentiating C2C12 cells. C2C12 cells with empty vector (top) or overexpressing cdk2-WT (middle) or cdk2-AF (bottom) were induced to differentiate. The rest are as described in Figure 1A. Primary FACS results are in Supplemental Figure S1. (D) Cell lysates from C were immunoprecipitated with indicated antibodies, and in vitro histone H1-kinase activities were measured. The phospho-H1 autoradiogram is shown in the top three panels, whereas 5% of input lysate immunoblotted with anti-cdk2 antibody is shown in the bottom panels. (E) Western blot analyses were performed for p21, p27, and cyclin A on the same cell lysates described in C and D. (F) Cell lysates from C were immunoblotted for differentiation markers myogenin and MHC. β-Actin and tubulin serve as loading controls.

Thus, the decrease in Wee1 kinase masked the increase of Y15 phosphorylation that would have been expected from Cdc25A decrease. The question then is whether the persistence of Y15 phosphorylation generated by Cdc25A loss is important for differentiation. To answer this, we created C2C12 cells stably overexpressing wild-type cdk2 (cdk2-WT) or cdk2 mutated as T14A+Y15F (cdk2-AF). Cdk2-AF will not show the persistent phosphorylation onT14/Y15 and so will not be inhibited by Cdc25A loss. For comparison, we used C2C12 cells infected with the empty retroviral vector (vector). On serum depletion, the vector-infected or the cdk2-WT–infected C2C12 cells showed an increase in G1 content and decrease in the other phases of the cell cycle (Figure 6C and Supplemental Figure S1, E–G). Overexpression of cdk2-AF, however, significantly impaired the G1 accumulation, strongly suggesting that phosphorylation of cdk2 on T14 and Y15 is essential for the cell cycle quiescence during muscle differentiation. As expected, the kinase activity of cdk2, cyclin A-associated cdks or cyclin B-associated cdks decreases rapidly upon serum depletion of vector or cdk2-WT infected C2C12 (Figure 6D). In contrast, the overexpression of cdk2-AF resulted in significant persistence of histone H1 kinase activity in all three immunoprecipitates all the way to day 3 of serum depletion. Moreover, Rb, a substrate for cdk2, remained phosphorylated in cells expressing Cdk2-AF compared to vector alone or Cdk2-WT–expressing cells upon myoblast to myotube differentiation (Supplemental Figure S8). The kinase activity persisted despite the induction of the cdk inhibitor p21 and the decrease in cyclin A (Figure 6E), showing that although other mechanisms for inhibiting the kinase were active in the differentiating C2C12 cells, phosphorylation of cdk2 on T14 and Y15 is a critical event for kinase inhibition. Thus, cdk2 has to be phosphorylated on T14 and Y15 for optimal G1 block in differentiating myoblasts.

The overexpression of cdk2-AF, but not cdk2-WT, also delayed the appearance of muscle-specific differentiation markers myogenin and MHC (Figure 6F). Consistent with the expectation that inhibition of Cdk2 will promote differentiation, acute inhibition of Cdk2 by roscovitine promotes C2C12 differentiation (Supplemental Figure S9). Thus persistent phosphorylation of cdk2 on T14 and Y15 due to the down-regulation of Cdc25A and resulting Cdk2 inhibition is important for optimal differentiation of myoblasts to myotubes.

Accumulation of Cancer Cells in G1 upon Transfection of miR-424 or -503 Also Requires Phosphorylation of cdk2 on T14 and Y15

Finally, we went back to the heterologous HCT116 colon cancer cells to test whether here, too, the G1 block created by these two microRNAs was dependent on the inhibitory phosphorylation of cdk2 on T14 and Y15. HCT116 cells infected with empty vector, or viruses overexpressing cdk2-WT or cdk2-AF were transfected with the two microRNAs, and the change in cell cycle distribution was measured relative to the transfection of a negative control siGL2 duplex (Figure 7A and Supplemental Figure S1, H–J). Both microRNAs increased the G1 population and decreased the S and G2 population of cells in the vector-infected or cdk2-WT–overexpressing C2C12. In contrast, overexpression of cdk2-AF diminished the G1 accumulation after transfection of miR-424 or -503. Thus, in the heterologous system, too, phosphorylation of cdk2 on T14 and Y15 is important for the G1 accumulation induced by the two microRNAs, consistent with our suggestion that Cdc25A is an important target for these microRNAs for their effect on the cell cycle.

Figure 7.

Accumulation of cancer cells in G1 upon transfection of miR-424 or -503 also requires phosphorylation of cdk2 on T14 and Y15; schematic of Cdc25A down-regulation during differentiation. (A) HCT116 cells expressing cdk2-AF were resistant to G1 arrest induced by miR-424 or -503. Cells stably infected with empty vector or viruses containing cdk2-WT or cdk2-AF were transfected either with control siRNA or miR-424 or si-503. The percentage of difference in miR-424 or -503–transfected cells in the indicated part of the cell cycle relative to control siRNA treated cells at 72 h is shown. Data are mean ± SD of least three independent experiments. Primary FACS results are in Supplemental Figure S1. (B) Cdc25A is down-regulated in differentiating myoblasts at the transcriptional, posttranscriptional and protein levels. The induced microRNAs miR-424 and -503 down-regulated Cdc25A protein synthesis, whereas existing protein is targeted to proteasomes by polyubiquitination-dependent pathways. The decrease in cdk2 kinase activity (which is facilitated by the concurrent induction of cdk2 inhibitors that is not shown) leads to E2F repression and repression of transcription from the Cdc25A promoter.